Phytate, the salt form of phytic acid (myo-inositol 1,2,3,4,5,6-hexakis dihydrogen phosphate), accounts for over 80% of total phosphorus in cereals and legumes, which, together with oilseed crops, are grown on over 90% of the world's harvested area (Reddy N. R., Pierson M. D., Sathe S. K. and Salunkhe D. K., 1989, Phytases in legumes and cereals. CRC Press, Inc., Boca Raton, Fla.). Although phytate is a storage form of phosphorus, phosphorus is not readily available to animals or plants, as a specific enzyme is required for the hydrolysis of phytate into inorganic phosphate.
Phytase, the enzyme that prefers phytate as its substrate, increases the availability of utilizable phosphorus by catalyzing the conversion of phytate into inorganic phosphate and myo-inositol phosphate and releasing phosphate to be utilized by animals and plants.
Over-expression of the phytase enzyme has been a long term and competitive topic in the biotechnology and enzyme production industry, due to the economical and environmental importance of the enzyme. Researchers have found ways to over-express the enzyme with the highest activity and the least number of purification steps to be carried out. Earlier studies on phytase expression were concerned with the extraction and production of the enzyme from fungal sources, which, until now, have been the only known source of for animal feed.
As early as in the 1980s, phytase was expressed in the extracellular medium from Aspergillus ficuum/niger (Ullah A. H. and Cummins B. J., 1988, Aspergillus ficuum extracellular pH 6.0 optimum acid phosphatase: purification, N-terminal amino acid sequence, and biochemical characterization. Preparative Biochemistry, 18(1):37-65). The enzyme was broadly studied by Ullah et al. in the same year. Until now, phytase from A. niger has been the most important commercial phytase. In the 1990s, the production of the enzyme was improved by a new biotechnology, i.e., expressing a recombinant protein in foreign strains, which was found promising in improving the yield of heterologous proteins. Fungal strains including Fusarium venenatum (Berka, R. M., Rey M. W, Brown K. M., Byun T, and Klotz A. V., 1998, Molecular characterization and expression of a phytase gene from the thermophilic fungus Fusarium venenatum. Applied and Environmental Microbiology, 64(11):4423-4427), Aspergillus niger and other Aspergillus species (Pasamontes L, Haiker M, Wyss M, Tessier M, and Loon A. P. G., 1997, Gene cloning, purification, and characterization of a heat-stable phytase from the fungus Aspergillus fumigatus. Applied and Environmental Microbiology, 63(5): 1696-1700; U.S. Pat. No. 5,830,733; U.S. Pat. No. 5,436,156; and U.S. Pat. No. 6,153,418); Klebsiella terrigena (Greiner R., Haller, E., Konietzny U., and Jany K. D., 1997, Purification and characterization of a phytase from Klebsiella terrigena. Archives of Biochemistry and Biophysics, 341(2):201-206); Thermomyces species (U.S. Pat. No. 5,866,118); and Schwanniomyces occidentalis (U.S. Pat. No. 5,840,561) have been reported to express heterogeneous phytase in significant amounts with appreciable activities. Many attempts to enzymatically hydrolyze phytate have been made which resulted in moderate improvements to the nutritional value of feed and a decrease in the amount of phosphorus excreted by animals, an environment benefit (Pen J., Verwoerd T. C., and Hoekema A., 1993, Phytase-containing transgenic seeds as novel feed additive for improved phosphorus utilization. Bio/Technology, 11:811-814).
While the enzyme production in fungi continues, other research groups have moved their focus to expressing phytase in yeast (Mayer A. F., Hellmuth, K., Schlieker H., Ulibarri R. L., Oertel S., Dahlems U., Strasser A. W. M. Strasser, and Loon A. P. G. M., 1998, An expression system matures: A highly efficient and cost-effective process for phytase production by recombinant strains of Hansenula polymorpha. Biotechnology and Bioengineering, 63(3):373-381; Han Y., Wilson, D. B., and Lei X. G., 1999, Expression of an Asperigillus niger phytase gene (phyA) in Saccharomyces cerevisiae. Applied and Environmental Microbiology. 65(5):1915-1918; Han Y and Lei X. G., 1999, Role of glycosylation in the functional expression of an Asperigillus phytase (phyA) in Pichia pastoris. Archives of Biochemistry and Biophysics, 364(1):83-90; Rodriguez E., Mullaney E. J., and Lei X. G., 2000, Expression of the Aspergillus fumigatus phytase gene in Pichia pastoris and characterization of the recombinant enzyme. Biochemical and Biophysical Research Communications, 268:373-378), plants (Ullah A. H. J., Sethumadhavan K., Mullaney E. J., 1999, Characterization of recombinant fungal phytase (phyA) expressed in tobacco leaves. Biochemical and Biophysical Research Communications, 264:201-206), and the enteric bacteria Escherichia coli (E. coli) (Dassa J., Marck C. and Boquet P. L., 1990, The complete Nucleotide Sequence of the Escherichia coli gene appA reveals significant homology between pH 2.5 acid phosphatase and glucose-1-phosphtase. Journal of Bacteriology, 172(9):5497-5500; Ostanin K., Harms E. H., Stevis P. E., Kuciel R., Zhou M. M., and Van Etten R. L., 1992, Overexpression, site-directed mutagenesis, and mechanism of Escherichia coli acid phosphatase. Journal of Bacteriology, 267(32):22830-22836; and Rodriguez E., Han Y. and Lei X. G., 1999, Cloning, Sequencing, and Expression of an Escherichia coli Acid Phosphatase/Phytase Gene (appA2) Isolated from Pig Colon. Biochemical and Biophysical Research Communications, 257:117-123). Other phytase sources from plants (Maugenest S., Martinez I and Lescure A, 1997, Cloning and characterization of a cDNA encoding a maize seedling phytase. Biochemistry Journal, 322:511-517) and mammals (Craxton A., Caffrey J. J., Burkhart W., Safrany S. T., and Shears S. B., 1997, Molecular cloning and expression of a rat hepatic multiple inositol polyphosphate phosphatase. Biochemistry Journal, 328:75-81) were also studied.
Several phytase genes in E. coli and Lactobacillus including EcAP (Ostanin K., Harms E. H., Stevis P. E., Kuciel R., Zhou M. M., and Van Etten R. L., 1992, Overexpression, site-directed mutagenesis, and mechanism of Escherichia coli acid phosphatase. Journal of Bacteriology, 267(32):22830-22836), appA (Dassa J., Marck C. and Boquet P. L., 1990, The complete Nucleotide Sequence of the Escherichia coli gene appA reveals significant homology between pH 2.5 acid phosphatase and glucose-1-phosphatase. Journal of Bacteriology, 172(9):5497-5500), appA2 (Rodriguez E., Han Y. and Lei X. G., 1999, Cloning, Sequencing, and Expression of an Escherichia coli Acid Phosphatase/Phytase Gene (appA2) Isolated from Pig Colon. Biochemical and Biophysical Research Communications, 257:117-123), and Lactobacillus plantarum (Zamudio et al., 2001, Lactobacillus plantarum phytase activity is due to non-specific acid phosphatase, Lett. App. Microbiol. 32:181-184), were identified and all were characterized as acid phosphatases with optimal enzyme activity at pH lower than 6.0. Other E. coli-derived phytases are disclosed in U.S. Pat. Nos. 6,183,740 and 6,190,897.
Although fungal and E. coli phytases have been expressed to significant amounts, the purification procedures for these phytases have been shown to be complicated and, in addition, these heterologously expressed enzymes often do not fold properly. For example, E. coli was found unable to express an active phytase enzyme originating from A. niger, because E. coli produces a non-glycosylated, intracellular inclusion protein that has a large molecular weight (Phillippy B. Q. and Mullaney E. J., 1997, Expression of an Aspergillus niger phytase (phyA) in Escherichia coli. Journal of Agricultural Food Chemistry, 45:3337-3342). Moreover, E. coli is an enteric bacterium that carries a risk of infecting animal gastro-intestinal tracts.
Several Bacillus strains are known to be GRAS bacterial strains. Genes encoding phytases have been cloned from Bacillus subtilis strains, VTT E-68013 (phyC; Kerovuo J., Laurarus M., Nurminen P., Kalkkinen N., and Apajalahti J., 1998, Isolation, characterization, molecular gene cloning, and sequencing of a novel phytase from Bacillus subtilis. Applied and Environmental Microbiology, 64(6):2079-2085, which is hereby incorporated by reference in its entirety) and DS11 (phyK; Kim Y. O., Lee J. K., Kim H. K., Yu J. H., and Oh T. K., 1998, Cloning of the thermostable phytase gene (phy) from Bacillus sp. DS11 and its overexpression in Escherichia coli, FEMS Microbiology Letters, 162:182-191; and U.S. Pat. No. 6,255,098, which are hereby incorporated by reference in their entireties). These reports showed characteristic differences of Bacillus phytases from fungal, E. coli, plant, and mammal phytases in that Bacillus phytases do not possess the conserved RHGXRXP domain sequence that are found in known phytases (Kerovuo et al., 1998, supra; Kim et al., 1998, supra). In addition, phytases from B. subtilis have been shown to have specific calcium dependence for its activity and thermostability (Kerovuo et al., 2000, The metal dependence of Bacillus subtilis phytase, Biochem. Biophys. Res. Commun. 268:365-369, which is hereby incorporated by reference in its entirety), which is not found in any other reported phytases from fungi, E. coli, plants and mammals. Furthermore, the pH optima for Bacillus subtilis phytase activity also differ from those of fungal and E. coli phytases. Many reports have demonstrated that the fungal as well as E. coli phytases are acid phosphatases with pH optima ranging from 2.5 (Rodriguez et al., 1999, supra; and Dassa et al., 1990, supra) to 5.5 (Han et al., 1999, supra). In contrast, the pH optima for Bacillus subtilis phytases are reported by Kerovuo et al. (1998, supra) to be 7. Thus, the phytase production using generally-regarded-as-safe (GRAS) bacterial strains has great utility as providing a new and safe source of phytase to be supplemented in commercial feeds.
Maugenest et al. (1997, Cloning and characterization of a cDNA encoding a maize seedling phytase, Biochemistry Journal 322:511-517) reported the cloning and characterization of a maize seedling phytase. U.S. Pat. No. 6,291,224 discloses a phytase derived from Zea mays and U.S. Pat. No. 6,303,766 discloses a phytase derived from soybean, both of which are known to be acidic phytases. However, in general, plant phytases are normally produced in insufficient amounts to suit industrial values, furthermore, in general, very low amounts of endogenous activity can be detected in non-germinated seeds. The extracellular phytase activity is obviously not significant enough for mobilizing phytate locked up in the soil.
Plants can obtain carbon, hydrogen and oxygen from water and photosynthesis, phosphorus, nitrogen, metal ions, calcium, and trace elements are mainly obtained from soil. Therefore, the availability of phosphorus and nitrogen in soil becomes a limiting factor for plant growth. Phosphorus, mainly in the form of inorganic phosphate, is absorbed from soil by roots and the inorganic phosphate will then be transported to the other tissues of the plant for various life processes, such as DNA and RNA synthesis, etc. However, the majority of phosphorus is locked up in plants, and stored in the form of phytate salts. For plants, the phosphorus locked up as phytate in the soil is not available for plant utilization. To supply plants with the nutritional needs, inorganic phosphate is commonly supplied in fertilizers to enhance plant growth, which constitutes another source of pollutant to the environment.
Efforts to express phytase in plants have not resulted in useful phenotypes. An acidic phytase from the fungus Aspergillus niger (phyA) was successfully expressed in transgenic tobacco (Ullah et al., 1999, supra). The recombinant phytase recovered from the transgenic tobacco was catalytically indistinguishable from the native phytase, except that the pH optima shifted from pH 5 to 4. The same gene was overexpressed in Arabidopsis (Richardson et al., 2000, Extracellular secretion of Aspergillus phytase from Arabidopsis roots enables plants to obtain phosphorus from phytate. Plant Journal 25(6):641-649). U.S. Pat. No. 6,022,846 discloses the expression of Aspergillus ficuum, Aspergillus niger, Aspergillus awamori, and Aspergillus ridulans, acidic phytases in the fruits, leaves, and roots of various crops, (also see U.S. Pat. No. 5,900,525). Intracellular expression of these acidic fungal phytases do not produce any significant phenotypic changes in the transformed plants.
Many monogastric animals, including pigs and chickens, were fed with feeds composed of soybean meal, corn, wheat, barley, rice bran and canola meal. Since most of the phosphorus is locked up in phytate salts, exogenous phytase enzymes with a low pH optimal, mainly from fungal origins, are frequently added as feed additives. Instead of adding exogenous phytases, incorporating transgenic plants expressing active phytases into animal feed will also enhance the availability of phosphate for animals fed with such feed. Thus, the need and desire continue to exist for methods which can affect and create biochemical pathways in plants through genetic engineering.